The present invention relates to inhibitors of blood vessel growth (angiogenesis) and particularly to amino acid sequences that can reduce or eliminate angiogenesis. In one aspect, the invention features methods for making angiogenesis inhibitors. The invention has a variety of applications including use in the treatment of angiogenesis-associated diseases such as cancer.
It is recognized that angiogenesis plays a role in diseases such as cancer. Therapies that can reduce or eliminate angiogenesis have been reported to be useful in the treatment of cancer and other angiogenesis-related diseases. See generally, Folkman J., (1971) N. Engl. Jour. Med. 285:1182; Folkman, J. (1989) J. Natl. Cancer Inst. 82: 4; O""Reilly, M. S. et al. (1994) Cell, 79: 315 and references cited therein.
There has been significant effort toward developing therapies that can reduce or eliminate angiogenesis. One approach has been to identify specific compounds that block or reduce growth of new blood vessels (neovascularization). See e.g., Ingber D, et al. (1990) Nature, 48:555; Clapp, C. et al. (1993) Endocrinology 133: 1292; and Folkman, J. (1995) N. Engl. J Med. 333: 1757.
Particular attention has focused on a compound called xe2x80x9cangiostatinxe2x80x9d. This compound has been reported to be a 38 kDa to 45 kDa fragment from plasminogen. See generally O""Reilly, M. S. (1997) in: Regulation of Angiogenesis (I D Goldberg and E M Rosen, eds) Birkhauser Velag (Basel, Switzerland), pgs. 273-294.
Plasminogen is a protein involved in many physiological processes such as clot lysis. The protein includes five linked domains that are sometimes called xe2x80x9ckringlesxe2x80x9d. Each kringle can be referred to as K1, K2, K3, K4 and K5, respectively. The K5 kringle is fused to a plasmin domain to form what is sometimes called a B chain. See e.g., Kelm R. J. et al (1994) J. Biol. Chem. 269:30147 and references cited therein.
Angiostatin has been disclosed as consisting of plasminogen kringles K1-4 Nucleic acid and protein sequences for angiostatin and various plasminogens have been reported. See e.g., Cao, Y. (1996) J. Biol. Chem. 271:29461; O""Reilley, M. S. et al. (1994, 1997) supra; and U.S. Pat No. 5,639,725.
Specific fragments of angiostatin have been reported to have anti-angiogenic activity. Sometimes the fragments are referred to as xe2x80x9cangiostatin-likexe2x80x9d to denote capacity to block blood vessel growth. Examples of such fragments include the K1and K1-3 Kringles. The K5 fragment has been reported to have minimal anti-angiogenic activity. See e.g., Cao, Y. supra.
There is increasing recognition that angiostatin is a strong inhibitor of neovascularization in vitro and in vivo. Thus, there has been an emerging need to develop effective methods for producing angiostatin and angiostatin-like fragments.
Several methods for producing angiostatin use proteases to cleave plasminogen into specific fragments. For example, one reported way to make angiostatin involves treating plasminogen with a protease called elastase. The fragments produced by the elastase are subsequently purified by conventional techniques.
However, the prior methods for making angiostatin are associated with significant problems. For example, many proteases produce disrupted kringle domains. That is, the proteases cleave plasminogen at more than one site. As a specific example, elastase has been reported to produce not only K1-4 (angiostatin) but also the K1-3 and K4 fragments. Although these methods can produce some angiostatin, isolation of significant amounts of the protein can be complicated by the other kringles. More generally, production of multiple fragments from plasminogen can substantially reduce yields and make purification of angiostatin and angiostatin-like fragments more difficult.
Additionally, many of the prior methods do not always completely remove the K5 fragment, thereby reducing the activity of certain angiostatin-like fragments.
One attempt to remedy this problem has been to minimize plasminogen cleavage. For example, one specific attempt has tried to reduce the amount of elastase used to cleave plasminogen so that larger kringle fragments can be obtained. However, this approach suffers from several drawbacks. For example, the need to reduce the amount or activity of the elastase makes assays more error prone and resistant to standardization and quality control. Use of other proteases has given rise to related problems. These shortcomings can be magnified by a variety of parameters, particularly during attempts to scale-up the methods.
The general availability of plasminogen and related molecules has supported efforts to isolate angiostatin by methods that include proteolytic cleavage. However, other approaches have been used to make angiostatin, e.g., recombinant DNA techniques. Use of the recombinant DNA techniques can be frustrated however by inability of some host cells to express suitable quantities of soluble protein. Additionally, there may be resistance to using recombinant DNA products in some settings.
It would be desirable to have angiogenesis inhibitors and more effective methods for producing same. It would be particularly desirable to have effective methods for making angiogenesis inhibitors from plasminogen that reduce or eliminate disruption of K1-4.
The present invention relates to angiogenesis inhibitors and effective methods for producing same from plasminogen. In general, we have discovered that by treating plasminogen with a specific cobra protein it is possible to enhance isolation of the angiogenesis inhibitors from plasminogen while minimizing or eliminating disruption of the first four plasminogen kringles (K1-4). The present invention has a variety of useful applications including use in the treatment of angiogenesis-associated diseases such as cancer.
In one aspect, we have found that cobra venom, particularly from the spitting cobra (Naja Nigricollis Nigricollis, hereinafter Naja Nigricollis), includes a protein and particularly a protease that is especially useful for producing certain angiogenesis inhibitors. More particularly, we have found that a protease (hereinafter xe2x80x9cK-4 proteasexe2x80x9d) found in cobra venom specifically cleaves plasminogen at a single site near the K4 and K5 kringles, thereby isolating, in a single fragment, nearly all of the K1-4 fragment. Disruption of the K1-4 fragment is reduced or eliminated by use of the K-4 protease. Additionally, we have found that use of the K-4 protease can enhance activity of the present angiogenesis inhibitors by removing the K5 fragment therefrom. Practice of the present invention can enhance the preparation and use of the angiogenesis inhibitors by significantly boosting yields of nearly intact K1-4 fragment.
In contrast to the present invention, prior methods for making anti-angiogenic compounds from plasminogen often use methods that disrupt the K1-4 fragment and do not always efficiently remove K5 therefrom. Thus, yields of intact K1-4 are often decreased by the prior methods.
FIG. 1 provides a schematic representation of human plasminogen including the plasmin catalytic domain (catalytic domain), the first five plasminogen kringles (K1-5) attached to the plasmin domain (5B chain), and the Naja Nigricollis (K-4) protease cleavage site.
The present angiogenesis inhibitors can be made by one or a combination of different strategies in accord with the invention. In one approach, plasminogen or other suitable plasminogen-related molecule is treated with an amount of the K-4 protease sufficient to cleave the plasminogen or related molecule at a single specific site. As will be discussed, that site has been determined to be near the border between of the K4 and K5 fragments of plasminogen. More particularly, the K-4 protease has been found to cleave plasminogen specifically between amino acid position 451 and 452. Thus, unless specified otherwise, the angiogenesis inhibitors of the invention include a C-terminal amino acid that corresponds to the cleavage of plasminogen at or near amino acid position 451.
Although the present angiogenesis inhibitors preferably include a specific C-terminus left by the K-4 protease, the N-terminus thereon can vary depending on several parameters such as the specific inhibitor desired and intended use. Generally, at least one suitable protease, preferably different from the K-4 protease, is used to make the N-terminus. Suitable proteases for making the N-terminus include those that cleave the plasminogen or plasminogen-related molecule once or more than once and in some instances multiple proteases may be used to make a desired N-terminus. Particular angiogenesis inhibitors of this invention will have an N-terminal amino acid between about amino acid 1 and amino acid 400 (inclusive) of plasminogen. More particular angiogenesis inhibitors will have an N-terminus at or near the N-terminal border of the K1 fragment, more particularly, between about amino acid positions 50 and 80 (inclusive) of plasminogen. For some applications, a suitable protease or multiple proteases can be used to provide an N-terminus between about amino acid position 80 and 400 (inclusive) of plasminogen.
As will be appreciated from the foregoing, the N-terminus of a desired angiogenesis inhibitor will be impacted by the protease(s) selected to provide the N-terminus. As an illustration of the invention, the K-4 protease may be used to cleave plasminogen or a plasminogen-related fragment followed by cleavage with a suitable protease or proteases to generate the N-terminus. Alternatively, at least one suitable protease may be used to cleave the plasminogen or related molecule followed by treatment with the K-4 protease. The order in which the K-4 protease and the other protease(s) are used is typically not important so long as the desired angiogenesis inhibitor is obtained. More specific methods for preparing angiogenesis inhibitors are described below.
Accordingly, in one aspect, the present invention features an isolated angiogenesis inhibitor having a molecular weight of between about 40 kDa to 50 kDa as determined by reducing gel electrophoresis and having an amino acid sequence substantially similar to that of the amino acid sequence shown in FIG. 9 or 10 (SEQ ID NO. 2 or 3). Particular angiogenesis inhibitors of this invention will include one or more carbohydrate groups such as glycosyl groups linked to specific amino acids therein. Angiogenesis inhibitors that include at least one glycosyl group are sometimes referred to xe2x80x9cglycoformsxe2x80x9d to denote potential for differential migration under specific reducing gel electrophoresis. See the examples and discussion below.
The angiogenesis inhibitors of the present invention preferably exhibit significant anti-angiogenic activity in a recognized in vivo or in vitro model for measuring angiogenesis. Several suitable assays to detect and measure angiogenesis are known in the field. Specific assays for testing the angiogenesis inhibitors are described below.
Particular angiogenesis inhibitors of the invention exhibit good activity in an in vitro angiogenesis assay. For example, certain endothelial cell proliferation assays are usually preferred for testing the activity of the angiogenesis inhibitors. More particular angiogenesis inhibitors exhibit a half maximal inhibitory dose (ID50) of between about 10 nM and about 500 nM or less in specific endothelial cell proliferation assays described below.
Additional particular inhibitors are capable of inhibiting tumor growth in a recognized in vivo assay for measuring angiogenesis. For example, certain in vivo carcinoma assays such as a lung carcinoma assays are generally preferred although in some instances other in vivo assays may be used. Preferred angiogenesis inhibitors of this invention exhibit significant capacity to inhibit neovascularization and are useful for treating a disease associated with undesired angiogenesis such as cancer. A variety of cancers can be treated in accord with the invention, e.g., prostate cancer, breast cancer, colon cancer, and lung cancer.
Further provided by the invention are pharmaceutical compositions that include at lease one angiogenesis inhibitor having a molecular weight of between about 40 kDa to 50 kDa as determined by reducing gel electrophoresis, and having an amino acid sequence substantially similar to that of the amino acid sequence shown in FIGS. 9 or 10 (SEQ ID NO. 2 or 3). Preferred are sterile pharmaceutical compositions such as those described in more detail below that are well-suited for administration to animals and particularly primates such as humans.
Additionally provided by the present invention is a substantially pure preparation of an angiogenesis inhibitor having: 1) a molecular weight of between about 40 kDa to 50 kDa, and 2) a C-terminal proline residue preferably corresponding to the Pro451 residue of plasminogen. Preferred preparations are provided as pharmaceutical compositions and are generally sterile.
In another aspect, the invention features an isolated plasminogen fragment having a molecular weight of between about 50 kDa and 70 KDa and having an amino acid sequence substantially similar to that of the amino acid sequence shown in FIG. 8 (SEQ ID NO: 1). Particular plasminogen fragments include one or more carbohydrate groups such as glycosyl groups bound to specific amino acids therein and may be referred to as xe2x80x9cglycoformsxe2x80x9d. As will be discussed below, the plasminogen fragments have a variety of useful applications including use in the production of certain angiogenesis inhibitors of this invention and use as molecular weight markers for electrophoretic applications.
The invention also provides for a substantially pure preparation of an isolated plasminogen fragment having: 1) a molecular weight of between about 50 kDa to 70 kD, and 2) a C-terminal proline preferably corresponding to the Pro451 residue of plasminogen. Preferred preparations are provided as pharmaceutical compositions and are generally sterile.
As discussed, the present invention features methods for making a variety of angiogenesis inhibitors.
For example, in one aspect, there is provided a method for making an isolated angiogenesis inhibitor having a molecular weight of between about 40 kDa to 50 kDa as determined by reducing gel electrophoresis and having an amino acid sequence substantially similar to that of the amino acid sequence shown in FIG. 9 or 10 (SEQ ID NO. 2 or 3). In one embodiment, the method includes at least one and preferably all of the following steps:
a) contacting plasminogen with an amount of the K-4 venom protease sufficient to cleave the plasminogen into fragments comprising fragments having a molecular weight of between about 50 kDa to 70 kDa as determined by reducing gel electrophoresis,
b) contacting the fragments with an amount of at least one other (second) protease sufficient to cleave the fragments at between about amino acid positions 50 and 80 of the plasminogen; and
c) isolating the angiogenesis inhibitor from the plasminogen fragments.
The plasminogen used in the method will generally include at least one carbohydrate group and particularly a glycosyl group. In another embodiment, the method can be used with a plasminogen-related molecule instead of plasminogen.
It will be appreciated that fragments made by practice of the method having a molecular weight of between about 50 kDa to 70 kDa will have a C-terminal residue corresponding to amino acid position 451 of plasminogen. In most instances, that C-terminal residue will be proline, e.g., as when human or mouse plasminogen is used in the method.
As noted, the method includes use of at least one (second) protease to cleave the plasminogen fragments having a molecular weight of between about 50 kDa to 70 kDa. Choice of the second protease will be guided by the N-terminus desired. Suitable proteases preferably cleave the plasminogen fragments between about amino acid 50 and amino acid 80 (inclusive) of plasminogen. More particular proteases will be capable of generating an N-terminus at or near the N-terminal border of the first plasminogen kringle. The protease may specifically cleave the plasminogen fragments generated in the method at one or more than one site as needed.
Illustrative of proteases suitable for use in the method are certain proteases and that are capable of cleaving plasminogen between about amino acid positions 1 and 400 (inclusive) with between about amino acids 50 and 80 being generally preferred. Methods for testing whether a particular protease can cleave plasminogen at the specified amino acids are known and include treating plasminogen with the desired protease and separating any fragments produced thereby by reducing gel electrophoresis. Essentially any cleavage site can be readily determined by inspection following use of a suitable fragment visualization technique such as staining.
In an embodiment of the above-described method, the plasminogen fragments having a molecular weight of between about 50 kDa and 70 kDa are purified prior to digestion with the second protease. In a more specific embodiment, the method further includes between steps a) and b), applying the cleaved plasminogen fragments to a first chromatographic implementation and then eluting same from the first chromatographic implementation to obtained a purified fraction thereof.
In a more specific embodiment, the method further includes applying the eluted fragments to a second chromatographic implementation capable that is preferably capable of specifically binding the fragments having the molecular weight of between about 50 kDa to 70 kDa. The fragments are then eluted from the second chromatographic implementation to obtain a substantially pure fraction that includes the fragments.
In another aspect, the invention provides a method for making an isolated angiogenesis inhibitor having a molecular weight of between about 40 kDa to 50 kDa and having an amino acid sequence substantially similar to that of the amino acid sequence shown in FIG. 9 or 10 (SEQ ID NO. 2 or 3). In this embodiment, the method uses specific plasminogen fragments obtained by prior treatment of plasminogen with the K-4 protease. In one embodiment, the method includes at least one and preferably all of the following steps:
a) contacting plasminogen fragments treated with the K-4 protease and having a molecular weight of between about 50 kDa to 70 kD as determined by reducing gel electrophoresis and an amino acid sequence substantially similar to that of the amino acid sequence shown in FIG. 8 (SEQ ID NO: 1) with at least one protease sufficient to cleave the plasminogen fragments between about amino acids 50 and 80 of plasminogen; and
b) isolating the angiogenesis inhibitor from the cleaved plasminogen fragments.
The plasminogen fragments will usually include at least one carbohydrate group and particularly a glycosyl group. Suitable proteases that can be used in accord with the method include those discussed above.
In an embodiment of the method, the plasminogen fragments having a molecular weight of between about 50 kDa and 70 kDa can be purified prior to digestion with the protease. In a more specific embodiment, the method further includes
between steps a) and b), applying the cleaved plasminogen fragments to a first chromatographic implementation and eluting same from the first chromatographic implementation.
In a more specific embodiment, the method further includes applying the eluted fragments to a second chromatographic implementation capable of specifically binding the fragments having the molecular weight of between about 50 kDa to 70 kDa. The fragments are then eluted from the second chromatographic implementation to obtain a substantially pure fraction of the fragments.
In another aspect of the invention, there is provided a method for making an isolated angiogenesis inhibitor having a molecular weight of between about 40 kDa to 50 kDa as determined by reducing gel electrophoresis and having an amino acid sequence substantially similar to that of the amino acid sequence shown in FIG. 9 or 10 (SEQ ID NO. 2 or 3). In one embodiment, the method includes at least one and preferably all of the following steps:
a) contacting plasminogen with a first protease sufficient to produce plasminogen fragments having a cleavage site between about amino acids 50 and 80 of the plasminogen,
b) contacting the plasminogen fragments with an amount of the K-4 protease sufficient to cleave the plasminogen fragments into fragments having a molecular weight of between about 50 kDa to 70 kD; and
c) isolating the angiogenesis inhibitor from the cleaved plasminogen fragments.
In one embodiment of the method, the plasminogen includes at least one carbohydrate group and particularly a glycosyl group. Suitable first proteases that can be used with the method are described above.
In an other embodiment of the method, the plasminogen fragments having a molecular weight of between about 50 kDa and 70 kDa can be purified prior to isolating the angiogenesis inhibitor. In a more specific embodiment, the method further includes
between steps b) and c) applying the cleaved plasminogen fragments to a first chromatographic implementation and then eluting same from the first chromatographic implementation.
In a more specific embodiment, the method further includes applying the eluted fragments to a second chromatographic implementation capable of specifically binding the fragments having the molecular weight of between about 50 kDa to 70 kDa. The fragments are then eluted from the second chromatographic implementation to obtain a substantially pure fraction of the fragments.
In another aspect, the invention features a method for making an isolated plasminogen fragment having a molecular weight of between about 50 kDa to 70 kDa as determined by reducing gel electrophoresis and having an amino acid sequence substantially similar to that of the amino acid sequence shown in FIG. 8 (SEQ ID NO: 1). As noted, the plasminogen fragments are useful for making the angiogenesis inhibitors of this invention. In one embodiment, the method includes at least one and preferably all of the following steps.
a) contacting plasminogen with an amount of the K-4 protease sufficient to cleave the plasminogen into fragments comprising fragments having a molecular weight of between about 50 kDa to 70 kDa,
b) eluting the cleaved plasminogen fragments from a first chromatographic implementation; and
c) isolating the fragments having the molecular weight of between about 50 kDa and 70 kDa to make the plasminogen fragment.
In one embodiment of the method, the plasminogen fragment includes at least one carbohydrate group and particularly a glycosyl group.
In a specific embodiment, the method further includes between steps b) and c) applying the eluted fragments to a second chromatographic implementation capable of specifically binding the fragments having the molecular weight of between about 50 kDa to 70 kDa. The fragments are subsequently eluted from the second chromatographic implementation to obtain a substantially pure fraction of the fragments.
A variety of chromatographic implementations can be used to practice the methods, e.g., chromatographic columns such as xe2x80x9cspinxe2x80x9d columns, and devices employing high performance liquid chromatographic (HPLC) implementations. Nearly any chromatographic implementation can be employed so long as it is capable of isolating the fragments as desired. More specific examples of suitable chromatographic techniques are provided below.
In preferred embodiments of the present methods, the angiogenesis inhibitors are derived from mammalian plasminogen and particularly primate or murine plasminogen with human plasminogen being preferred for many applications.
The K-4 protease used in the present methods can be obtained by one or a combination of strategies. For example, in one approach, the protease is isolated from a fluid obtained from the spitting cobra and is usually purified therefrom to enhance specific activity. In a more specific approach, the protease is isolated from the venom of the cobra by conventional enzyme isolation techniques such as chromatography, filtration or the like. Preferred are isolation techniques that enhance activity of a venom fraction that is capable of specifically cleaving human plasminogen between amino acid positions Pro451 and Asn452 or Asp432 depending on sequence. As noted, the specific cleavage can be determined by a variety of assays including reducing gel electrophoresis.
A particularly preferred K-4 protease for use in accord with the invention is isolated from venom of the cobra and has a molecular weight of between about 5 kDa and 20 kDa and more typically about 10 kDa as determined by reducing gel electrophoresis. Especially preferred methods of isolating the Naja Nigricollis protease are described below.
Further provided by the present invention are methods for inhibiting tumor growth in a mammal such as a primate, rodent or domesticated animal. In one embodiment, the method includes administering a therapeutically effective amount of at least one angiogenesis inhibitor sufficient to inhibit the tumor growth. In a specific embodiment, the angiogenesis inhibitor has a molecular weight of between about 40 kDa to 50 kDa as determined by reducing gel electrophoresis and has an amino acid sequence substantially similar to that of the amino acid sequence shown in SEQ ID NOS: 1 or 4. The angiogenesis inhibitor may be administered alone or in combination with other therapeutic agents, e.g., anti-angiogenic compounds or other anti-tumor agents such as chemotherapeutic drugs. Preferably, the angiogenesis inhibitor is administered as a part of a pharmaceutical composition and is sterile.
In a specific embodiment of the method, the mammal is a human and particularly a patient suffering from or suspected of suffering from tumor growth. In a more specific embodiment, the angiogenesis inhibitor is derived from human plasminogen.
Further aspects of the present invention are discussed infra.